Genetic Engineering: Cloning and Restriction Modification

1. Unit II Lecture 1 B. Tech. (Biotechnology) III Year V th Semester EBT-501, Genetic Engineering

2. EBT 501, Genetic Engineering Unit I Gene cloning -concept and basic steps; application of bacteria and viruses in genetic engineering; Molecular biology of E. coli and bacteriophages in the context of their use in genetic engineering, Cloning vectors: Plasmid cloning vector PBR322, Vectors for cloning large piece of DNA; –Bacteriophage-l and other phage vectors; Cosmids, Phagemids; YAC and BAC vectors, Model vectors for eukaryotes – Viruses, Unit II Restriction modification, enzymes used in recombinant DNA technology endonucleases, ligases and other enzymes useful in gene cloning, PCR technology for gene/DNA detection, cDNA, Use of Agrobacterium for genetic engineering in plants; Gene libraries; Use of marker genes. Cloning of foreign genes: DNA delivery methods -physical methods and biological methods, Genetic transformation of prokaryotes: Transferring DNA into E. coli –Chemical induction and Electroporation, Unit III Gene library: Construction cDNA library and genomic library, Screening of gene libraries – screening by DNA hybridization, immunological assay and protein activity, Marker genes: Selectable markers and Screenable markers, nonantibiotic markers, Gene expression in prokaryotes: Tissue specific promoter, wound inducible promoters, Strong and regulatable promoters; increasing protein production; Fusion proteins; Translation expression vectors; DNA integration into bacterial genome; Increasing secretions; Metabolic load, Recombinant protein production in yeast: Saccharomycescerevisiae expression systems; Mammalian cell expression vectors: Selectable markers; Unit IV Origins of organismal cloning in developmental biology research on frogs; nuclear transfer procedures and the cloning of sheep (Dolly) & other mammals; applications in conservation; therapeutic vs. reproductive cloning; ethical issues and the prospects for human cloning; Two-vector expression system; two-gene expression vector, Directed mutagenesis; transposon mutagenesis, Gene targeting, Site specific recombination Unit V General principles of cell signaling, Extracellular signal molecule and their receptors, Operation of signaling molecules over various distances, Sharing of signal information, Cellular response to specific combinations of extracellular signal molecules; Different response by different cells to same extracellular signal molecule, NO signaling by binding to an enzyme inside target cell, Nuclear receptor; Ion channel linked, G-protein- linked and enzyme-linked receptors, Relay of signal by activated cell surface receptors via intracellular signaling proteins, Intracellular signaling proteins as molecular switches, Interaction between modular binding domain and signaling proteins, Remembering the effect of some signal by cells.

3. Restriction ModificationUnderstandingBASIC BIOLOGY

4. Understanding the biological basis of host controlledrestriction and modification of bacteriophage DNA led to the identification of restriction endonucleases • Before 1970 there was no method for cleaving DNA at discrete points. All the available methods for fragmenting DNA were non-specific. • The available endonucleases had little site specificity and chemical methods produced very small fragments of DNA • Meselson and Yuan (1968) Purified restriction endonuclease of Escherichia coli K12 for the first time • While the enzyme does bind to a defined recognition sequence, cleavage occurs at a “random” site several kilobases away

5. In 1970 Kelly & Smith with the discovery in Hemophilus influenzae • the enzyme recognizes a particular target sequence in a duplex DNA molecule and breaks the polynucleotide chain within that sequence • Restriction systems allow bacteria to monitor the origin of incoming DNA and to destroy it if it is recognized as foreign. • Restriction endonucleases recognize specific sequences in the incoming DNA and cleave the DNA into fragments, either at specific sites or more randomly.

6. When the incoming DNA is a bacteriophage genome, the effect is to reduce the efficiency of plating, i.e. to reduce the number of plaques formed in plating tests. • The phenomena of restriction and modification were well illustrated and studied by the behavior of phage lamda on two E. coli host strains.

7. If a stock preparation of phage λ, for example, is • made by growth upon E. coli strain C and this stock • is then titered upon E. coli C and E. coli K, the titers • observed on these two strains will differ by several • orders of magnitude • the titer on E. coli K being the lower. The phage are said to be restricted by the second host strain (E. coli K). • When those phage that do result from the infection of E. coli K are now replicated on E. coli K they are no longer restricted; but if they are first cycled through E. coli C they are once again restricted when plated upon E. coli K (Fig.) • Thus the efficiency with which phage λ plates upon a particular host strain depends upon the strain on which • it was last propagated.

8. This non-heritable change conferred upon the phage by the second host strain explains why phage that survive one cycle of growth upon the restrictive host can subsequently reinfect that host efficiently • their DNA has been replicated in the presence of the modifying methylase and so it, like the host DNA, becomes methylated and protected from the restriction system.

10. Four different types of restriction and • modification (R-M) system have been • recognized but only one is widely used in • gene manipulation

11. Most of the useful R-M systems are of type II. • advantages over type I and III systems • restriction and modification are mediated by separate enzymes so it is possible to cleave DNA in the absence of modification • restriction activities do not require cofactors such as ATP or S-adenosylmethionine, making them easier to use • type II enzymes recognize a defined, usually symmetrical, sequence and cut within it. Many of them also make a staggered

14. Construction of Recombinant DNA Molecules In Vitro